The present invention relates to biosensors, and in particular, to a biosensor for measuring a binding event between a ligand and a ligand-binding receptor, and to methods for producing such biosensors.
Diagnostic tools used for detecting or quantitating biological analytes typically rely on ligand-specific binding between a ligand and a receptor. Ligand/receptor binding pairs used commonly in diagnostics include antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, substrate/enzyme, and complementary nucleic acid strands. The analyte to be detected may be either member of the binding pair; alternatively, the analyte may be a ligand analog that competes with the ligand for binding to the complement receptor.
A variety of devices for detecting ligand/receptor interactions are known. The most basic of these are purely chemical/enzymatic assays in which the presence or amount of analyte is detected by measuring or quantitating a detectable reaction product, such as gold immunoparticles. Ligand/receptor interactions can also be detected and quantitated by radiolabel assays.
Quantitative binding assays of this type involve two separate components: a reaction substrate, e.g., a solid-phase test strip and a separate reader or detector device, such as a scintillation counter or spectrophotometer. The substrate is generally unsuited to multiple assays, or to miniaturization, for handling multiple analyte assays from a small amount of body-fluid sample.
In biosensor diagnostic devices, by contrast, the assay substrate and detector surface are integrated into a single device. One general type of biosensor employs an electrode surface in combination with current or impedance measuring elements for detecting a change in current or impedance in response to the presence of a ligand-receptor binding event. This type of biosensor is disclosed, for example, in U.S. Pat. No. 5,567,301.
Gravimetric biosensors employ a piezoelectric crystal to generate a surface acoustic wave whose frequency, wavelength and/or resonance state are sensitive to surface mass on the crystal surface. The shift in acoustic wave properties is therefore indicative of a change in surface mass, e.g., due to a ligand-receptor binding event. U.S. Pat. Nos. 5,478,756 and 4,789,804 describe gravimetric biosensors of this type.
Biosensors based on surface plasmon resonance (SPR) effects have also been proposed, for example, in U.S. Pat. Nos. 5,485,277 and 5,492,840. These devices exploit the shift in SPR surface reflection angle that occurs with perturbations, e.g., binding events, at the SPR interface. Finally, a variety of biosensors that utilize changes in optical properties at a biosensor surface are known, e.g., U.S. Pat. No. 5,268,305.
Biosensors have a number of potential advantages over binding assay systems having separate reaction substrates and reader devices. One important advantage is the ability to manufacture small-scale, but highly reproducible, biosensor units using microchip manufacturing methods, as described, for example, in U.S. Pat. Nos. 5,200,051 and 5,212,050.
Another advantage is the potentially large number of different analyte detection regions that can be integrated into a single biosensor unit, allowing sensitive detection of several analytes with a very small amount of body-fluid sample. Both of these advantages can lead to substantial cost-per-test savings.
A key element in the manufacture of biosensors, particularly multi-assay biosensors, is the placement of analyte-specific binding molecules or enzymes at desired locations on a biosensor surface. Ideally, it would be desirable to construct a universal biosensor surface under rigorous microchip manufacturing conditions, but allow a variety of different surface-region formats to be achieved under less restrictive manufacturing conditions, which at one extreme would allow an end user to tailor the universal chip to a unique multi-analyte format.
In one aspect, the invention includes a biosensor apparatus for detecting a binding event between a ligand and ligand-binding agent. The apparatus has a biosensor surface, and two-subunit heterodimer complexes carried on the surface. The complexes are composed of first and second, preferably oppositely charged peptides that together form an xcex1-helical coiled-coil heterodimer. The first peptide is attached to the biosensor surface, and a ligand is covalently attached to the second peptide, accessible for binding by a ligand-binding agent. Binding of an anti-ligand agent to the ligand is detected by a suitable detector in the apparatus.
The first peptide subunit may be attached to the biosensor surface covalently, e.g., through an oligopeptide spacer or a hydrocarbon-chain spacer, or may be bound to the biosensor surface through a stable non-covalent linkage, e.g., a biotin/avidin binding pair. The biosensor surface may include multiple regions, each having a different selected ligand attached to the second-subunit peptide.
In one general embodiment, the biosensor surface includes a monolayer composed of hydrocarbon chains anchored at their proximal ends to the biosensor surface, and having free distal ends defining an exposed monolayer surface. The heterodimer complexes in this embodiment are preferably embedded in the monolayer, and the ligands are disposed on or near the monolayer surface. The monolayer may be formed on a metal, e.g., gold film, and may be composed of 8-22 carbon atom chains attached at their proximal ends to the biosensor surface by a thiol linkage. The chains have a preferred molecular density of about 3 to 5 chains/nm2, and the dielectric constant of the monolayer, in the presence of such solution but in the absence of such binding receptor, is preferably less than about 2.
In a biosensor apparatus designed for amperometric detection of binding of a ligand-binding agent to the monolayer ligand, the biosensor surface is an electrode, and the monolayer, including the heterodimer complexes embedded in the monolayer, is sufficiently close-packed and ordered to form an effective barrier to current across the monolayer mediated by a redox ion species in an aqueous solution in contact with the monolayer. Binding of a ligand-binding agent to the ligand on the monolayer surface is effective to increase current across the monolayer, mediated by such redox species. A chamber in the apparatus is adapted to contain an aqueous solution of redox species in contact with the monolayer, and the detector includes a circuit for measuring ion-mediated current across the monolayer, in response to binding events occurring between the receptor and ligand.
In a biosensor apparatus designed for gravimetric detection of binding of a ligand-binding agent to the surface-bound ligand, the biosensor surface is a piezoelectric crystal. The detector functions to (i) generate a surface acoustic wave in the crystal and (ii) detect the shift in wave frequency, velocity, or resonance frequency of the surface acoustic wave produced by binding of ligand-binding agent to the ligand.
In a biosensor designed for optical surface plasmon resonance (SPR) detection of binding of a ligand-binding agent to the surface-bound ligand, the biosensor surface is a transparent dielectric substrate coated with a thin metal layer on which the monolayer is formed, where the substrate and metal layer form a plasmon resonance interface. The detector functions to excite surface plasmons at a plasmon resonance angle that is dependent on the optical properties of the metal film and attached monolayer, and to detect the shift in plasmon resonance angle produced by binding of ligand-binding agent to the ligand.
In a biosensor designed for optical detection of binding of a ligand-binding agent to the surface bound ligand, the detector functions to irradiate the biosensor surface with a light beam, and detect a change in the optical properties of the surface layer, e.g., monolayer with embedded heterodimer, produced by binding of ligand-binding agent to the ligand.
In another aspect, the invention includes a method for producing a ligand-specific biosensor for use in a biosensor apparatus capable of detecting a binding event between a ligand and ligand-binding receptor. The method involves contacting together: (a) a biosensor electrode having a biosensor surface and a first heterodimer-subunit peptide attached to the biosensor surface, and (b) a second, preferably oppositely charged peptide capable of binding to the first peptide to form a two-subunit xcex1-helical coiled-coil heterodimer. The second peptide has an attached ligand capable of binding specifically to a ligand-specific agent. The contacting is effective attach ligands to the biosensor surface. The biosensor surface may include first and second discrete regions, where the second heterodimer subunit peptide in each region has a different attached ligand.
In one general embodiment of the method, the biosensor surface has a monolayer composed of hydrocarbon chains (i) anchored at their proximal ends to the biosensor surface, and (ii) having free distal ends defining an exposed monolayer surface. The first peptide is embedded in the monolayer, and binding of the second peptide to surface-bound first peptide is effective to dispose the ligand preferably on or near the monolayer surface.
More generally, the invention provides a method of constructing an array of different, selected biological reagents attached to different, selected regions on an assay support surface. The method includes attaching molecules of a first heterodimer-subunit peptide to the support surface, effective to cover the different regions on the surface with the first peptide molecules. The subunit peptide has protecting groups which when photo-released, allow the peptide to interact with a second, preferably oppositely charged heterodimer-subunit peptide, to form a two-subunit xcex1-helical coiled-coil heterodimer.
The surface is irradiated in a selected region of the surface under conditions effective to deprotect the first peptide in the irradiated region only, then contacted with a second subunit peptide carrying the assay reagent. This contacting is effective to attach the selected reagent to the exposed region of the surface only. The above steps are repeated for different selected regions and assay reagents, until the desired array of different, selected biological reagents disposed at different selected regions on an assay support surface is produced.
In one embodiment, the first subunit peptide contains amino acid residues with one or more protected carboxyl groups, e.g., glutamate groups with nitrophenolate protecting groups.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.